Kit and method for detecting an active substance

Abstract

The invention relates to a kit for detecting an active substance, which is an α1-adrenoceptor antagonist, in a sample, wherein the kit comprises: capture antibodies which are designed to bind to the active substance; primary antibodies which are designed to bind to the active substance while the active substance is bound to a capture antibody; and a detection means, wherein the detection means is associated with the primary antibody. The invention also relates to a method for detecting an active substance.

Description

[0001] 65387 / MB

[0002] Borkenstein & Borkenstein Specialists in Ophthalmology OG, Kreuzgasse 35, 8010 Graz (AT)

[0003] Kit and method for detecting an active ingredient

[0004] The present invention relates, inter alia, to a kit and a method for detecting an active ingredient, as defined in the independent claims.

[0005] Cataracts (also known as senile cataracts) are a common eye disease, particularly in older patients, characterized by a clouding of the eye's lens, leading to a gradual impairment of vision. This condition is treated with cataract surgery, in which the existing lens is removed and replaced with an artificial one. Approximately 37 million such operations are performed worldwide each year.

[0006] Even though cataract surgeries using modern techniques are usually uncomplicated today and generally adhere to a high safety standard, undesirable events or complications can occur in some cases. One of these is the so-called Intraoperative Floppy Iris Syndrome (IFIS). IFIS is characterized by the following triad: 65387 / MB Borkenstein & Borkenstein Specialists in Ophthalmology OG, Kreuzgasse 35, 8010 Graz (AT)

[0007] - Undulating iris: The iris moves despite the administration of mydriatics even with small intraoperative fluid flows.

[0008] - Iris prolapse: The iris prolapses through the surgical access points to the anterior chamber (despite proper incision), which can result in iris defects.

[0009] - Intraoperative progressive miosis; despite application of mydriatics, there is no further pupil dilation but rather pupil constriction.

[0010] While the iris should remain "wide-open" throughout the entire course of an operation to allow a good view of the actual surgical field (lens, capsule), in IFIS, constrictions or fluctuations in the size of the iris occur during the operation, which can be particularly problematic during phacoemulsification, i.e., the removal of the existing lens.

[0011] Complications that can arise from IFIS are numerous and include, among others: iris injuries; a distorted pupil with expected increased glare sensitivity and / or dysphotopsia, halo or glare symptoms, and reduced visual acuity; posterior capsule rupture; retinal detachment; and loss of lens fragments into the vitreous cavity. In summary, IFIS can lead to a poorer surgical outcome and impaired vision. The risk of complications is further increased, particularly in less experienced surgeons.

[0012] According to studies, the overall incidence of IFIS is between 5 and 10%, although evaluations have also shown that in men over 80 years of age, the IFIS incidence can be as high as 80-90%.

[0013] It has been found that the occurrence of IFIS is associated with the concurrent or prior use of pharmaceutical agents that are cd-adrenoceptor antagonists. Examples of such agents are tamsulosin, alfuzosin, and doxazosin. These are frequently used to treat benign prostatic hyperplasia (BPH). The target of these substances, the cd-adrenoceptor, contributes to the regulation of tone not only in the smooth muscle of the urinary tract but also in the dilator pupillae muscle. 65387 / MB Borkenstein & Borkenstein Specialists in Ophthalmology OG, Kreuzgasse 35, 8010 Graz (AT)

[0014] Blockage of the receptor leads to insufficient contraction of the dilator pupillae muscle and thus to inadequate dilation of the pupil.

[0015] Therefore, medications such as tamsulosin should either be discontinued as early as possible before cataract surgery, if this seems justifiable from a urological point of view, or the use of these medications should be reported and at least discussed with the surgeon.

[0016] However, it is known that patients with urological complaints often do not disclose them to an ophthalmologist, primarily for reasons of privacy, and consequently, they also fail to disclose their medication with the aforementioned drugs. This results in a high number of undiagnosed patients taking α1-adrenoceptor antagonists.

[0017] It is assumed that reducing this number of unreported cases will reduce the incidence of IFIS, especially in older male patients, and generally the frequency of adverse events and / or complications.

[0018] One object of the present invention can therefore be seen as preventing the occurrence of Intraoperative Floppy Iris Syndrome (IFIS) during cataract surgery.

[0019] To solve these and possibly other problems, a test or assay is now proposed that enables the rapid and uncomplicated detection of the presence of an α1-adrenoceptor antagonist in a sample.

[0020] This could potentially reduce not only the aforementioned number of unreported cases of patients with unknown α1-adrenoceptor antagonist use, but also allow for the targeted postponement of surgeries or the implementation of additional safety measures if the α1-adrenoceptor antagonist level has not yet decreased sufficiently despite discontinuation of the medication. For example, it would be conceivable to administer additional or higher doses of pupil-dilating medication (e.g., cyclopentolate, atropine, neosynephrine) before a planned / elective surgery, or to use additional aids / devices such as iris retractors or iris retractor rings intraoperatively to maintain iris dilation (mydriasis) throughout the operation.

[0021] Preferably, this can be done using an antibody-based assay, wherein the assay uses or contains antibodies that are specific for the α1-adrenoceptor antagonist under investigation.

[0022] The assay can be designed in different ways and may, for example, be a sandwich assay and / or an electrochemical assay. Generally, the terms "assay," "kit," "detection kit," and the like are used interchangeably here unless the context clearly indicates otherwise.

[0023] The assay may include an immobilized antibody specific to the drug. This antibody, also known as a capture antibody, can be immobilized on a solid support, such as a particulate or porous support. The capture antibody can be immobilized using linkers that bind the antibody to the support surface. Such supports are well-known in the field and include, for example, thiol-reactive, amine-reactive, and PEGylated linkers, as well as protein-based linkers, such as the streptavidin-biotin system.

[0024] The capture antibody is located, in particular, in a detection area of ​​the carrier. This detection area can be located at a distance from a sample delivery area, but it can also be located essentially in the same place as the sample delivery area.

[0025] If the assay is a sandwich assay, it may further include a primary antibody designed to bind specifically to the drug. The primary antibody may be substantially identical to the capture antibody in terms of its binding properties, and the primary antibody should be able to bind to the drug if the drug is already bound to the capture antibody. 65387 / MB

[0026] Borkenstein & Borkenstein Specialists in Ophthalmology OG, Kreuzgasse 35, 8010 Graz (AT)

[0027] Capture antibodies and primary antibodies can be the same or different antibodies.

[0028] Furthermore, the assay may include a detection agent, which can be configured according to the desired detection method. The detection method used by the assay described here can be, for example, colorimetric, fluorometric, luminometric, electrochemical, or radiometric, in order to produce a positive or negative result (i.e., the presence or absence of a detectable amount of active ingredient in the sample).

[0029] In one embodiment, the assay is a colorimetric assay in which the detection agent comprises a dye that is directly or indirectly bound to the primary antibody or can bind directly or indirectly to it.

[0030] In another embodiment, the assay is a colorimetric assay functioning like an ELISA (enzyme-linked immunosorbent assay). In this case, the detection agent may comprise an enzyme that is directly or indirectly bound to, or capable of binding to, the primary antibody. Furthermore, the detection agent may comprise a dye precursor that can be converted to a dye by a chemical or biochemical reaction catalyzed by the enzyme. The detection agent may additionally or alternatively generate an electrochemical signal.

[0031] The ELISA can be a competitive ELISA.

[0032] In the context of the assays described here, "directly bound" can mean that a molecule, in particular the dye or enzyme, is covalently bound to the primary antibody without the use of a linker or other binding agent. Conversely, "indirectly bound" can mean that a molecule, in particular the dye or enzyme, is bound to the primary antibody using a linker or other binding agent. The linker can be a linker molecule that establishes a covalent bond between the molecule and the primary antibody. The binding agent can also be a secondary antibody, which is designed to bind specifically to the primary antibody when the latter is bound to the drug. In this case, the molecule can be bound directly or indirectly to the secondary antibody.

[0033] In a preferred embodiment, the assay is a so-called rapid test in which a carrier is provided as a porous carrier, for example made of cellulose acetate, on which the capture antibody, i.e. a plurality of capture antibodies, is immobilized.

[0034] The capture antibody is located in a detection area, which is spaced apart from a sample delivery area. The detection area and the sample delivery area are in fluid-conducting communication, whereby liquid applied to the sample delivery area is transported into the detection area, particularly by capillary action.

[0035] In particular, the assay or kit may be an immunosorbent assay, for example in the form of an immunological test strip.

[0036] The carrier may also include a control area, spaced apart from the detection area, in which a target specific to the primary antibody is immobilized. The control area is also in fluid-conducting communication with the sample delivery area.

[0037] The rapid test or kit may further include a sample vial containing a primary antibody and a detection agent, the sample vial being designed to receive a sample. The sample vial may also include a buffer solution or another solvent suitable for the functionality of the assay.

[0038] Such an assay can be used in particular as follows: The sample is placed in the sample container and then applied, together with the primary antibody and detection agent, to the sample application area of ​​the carrier. The capillary action of the carrier transports the applied liquid towards the detection and control areas. Any substances contained in the sample are... 65387 / MB Borkenstein & Borkenstein Specialists in Ophthalmology OG, Kreuzgasse 35, 8010 Graz (AT)

[0039] Drug molecules are bound by capture antibodies in the detection area. Furthermore, the drug molecules are bound by primary antibodies, resulting in coupling with the detection agent, which can be a dye. The primary antibodies can be placed on the carrier, either alternatively or additionally, where they can bind to the drug molecules.

[0040] The presence of active ingredient molecules in the sample is made visible by a color change in the detection area.

[0041] The assay can enable a qualitative and / or quantitative analysis of the active ingredient.

[0042] The targets immobilized in the control region are also bound by the primary antibodies, resulting in coupling between the target and the detection agent. Therefore, a color change occurs in the control region, even if the sample contains no drug molecules.

[0043] The sample can be a bodily fluid from a subject who is being or has been treated with a drug containing an α1-adrenoceptor antagonist as its active ingredient. The sample can be, for example, blood, serum, or urine.

[0044] It should be noted that the term "antibody" is not necessarily to be understood as a complete antibody. Thus, an "antibody" within the meaning of this invention can also include antibody fragments or hybrid molecules derived from antibodies, as long as they possess the ability to bind specifically to the active substance.

[0045] In this context, "specifically binding" means, in particular, that the antibody has a significantly higher binding affinity towards the active substance than towards any other target.

[0046] The invention may relate to a kit for detecting an active ingredient that is an α1-adrenoceptor antagonist in a sample. 65387 / MB Borkenstein & Borkenstein Specialists in Ophthalmology OG, Kreuzgasse 35, 8010 Graz (AT)

[0047] The kit may optionally include: capture antibodies designed to bind to the active substance; primary antibodies designed to bind to the active substance while the active substance is bound to a capture antibody; and a detection agent, wherein the detection agent is associated with the primary antibody.

[0048] If applicable, the active ingredient is (R)-(-)-5-{2-[2-(2-Ethoxyphenoxy)- ethylamino]propyl}-2-methoxybenzenesulfonamide or a pharmaceutically acceptable compound or derivative thereof.

[0049] If necessary, the capture antibodies are immobilized on a solid support.

[0050] Optionally, the capture antibodies are provided to be immobilized on a detection area of ​​a solid support, with the detection area being in fluid-conducting communication with a sample introduction area.

[0051] Optionally, the support may consist of nanoparticles, in particular gold or silver nanoparticles, or may comprise or be formed from a polymer material, in particular a porous one.

[0052] Optionally, the detection agent may comprise an enzyme and a detection reagent, wherein the enzyme is bound to the primary antibody, wherein the detection reagent is configured to undergo a reaction in which at least one property of the detection reagent is altered, and wherein the reaction is catalyzed by the enzyme.

[0053] Optionally, the detection agent may comprise secondary antibodies, an enzyme, and a detection reagent, wherein the secondary antibodies are configured to bind to a primary antibody while the latter is bound to the active substance, wherein the enzyme is bound to the secondary antibody, wherein the detection reagent is configured to undergo a reaction in which at least one property of the detection reagent is altered, and wherein the reaction is catalyzed by the enzyme.

[0054] The kit may be an ELISA kit.

[0055] Optionally, the detection reagent is provided to be a dye precursor, and the property of the detection reagent that is changed during the reaction is the absorption coefficient for electromagnetic radiation in the visible and / or ultraviolet light range.

[0056] If necessary, the detection agent may include a dye that is bound to, or can bind to, the primary antibodies.

[0057] If necessary, the dye is intended to be colloidal gold.

[0058] Optionally, a control area is provided on the substrate, which is in fluid-conducting communication with the sample application area, wherein an immobilized control substance is provided in the control area, wherein the control substance is designed to be bound, in particular specifically, by the primary antibodies.

[0059] If appropriate, the sample is intended to be a body fluid, in particular urine, serum or blood, of a subject who is or has been treated with a drug containing the α-adrenoceptor antagonist.

[0060] If necessary, the primary antibodies and the detection agent are provided in one sample container.

[0061] The sample container may contain an aqueous buffer solution. 65387 / MB Borkenstein & Borkenstein Ophthalmologists OG, Kreuzgasse 35, 8010 Graz (AT)

[0062] If necessary, it is provided that the capture antibody and / or the primary antibody captures the active substance with a dissociation constant (Kd) of not more than 10⁻¹⁰. 6 M, in particular at most 10' 7 , preferably no more than 10' 8 binds.

[0063] If applicable, the capture antibody and / or the primary antibody is designed to bind selectively to the active substance and to exhibit no significant cross-reactivity with other active substances. In particular, cross-reactivity with β-adrenergic receptor antagonists, α2-receptor antagonists, antidepressants, and benzodiazepines may each be less than 2%.

[0064] If applicable, the capture antibody and / or the primary antibody is designed to bind a structural motif of the active substance, which may include, in particular, an aromatic group and / or a protonable amine function.

[0065] If necessary, the capture antibody and / or the primary antibody is designed to maintain binding to the active substance even in the presence of other pharmacologically active substances.

[0066] Optionally, the capture antibody and / or the primary antibody is provided for by immunizing a mammal with a hapten conjugate consisting of the active substance and a carrier protein, wherein the tamsulosin hapten in particular contains a functional group of tamsulosin coupled to a carrier protein via a C2-C8 alkyl spacer or a polyethylene glycol spacer.

[0067] If necessary, the active ingredient is selected from the group consisting of doxazosin, tamsulosin, alfuzosin, silodosin, and phenoxybenzamine. In particular, the active ingredient is tamsulosin.

[0068] The invention may relate to a method for detecting an active ingredient using a kit as described herein. 65387 / MB Borkenstein & Borkenstein Ophthalmologists OG, Kreuzgasse 35, 8010 Graz (AT)

[0069] If necessary, the procedure includes the following steps: collecting a sample; mixing the sample with at least the primary antibodies and the detection agent; reading out a signal indicating the presence of active substance in the sample.

[0070] Further features of the invention will become apparent from the claims, the description of the embodiment, and the figure. The invention is explained in detail below using a preferred embodiment as an example. This embodiment serves only to illustrate advantageous effects of the invention and is not intended to limit the scope of protection defined by the claims.

[0071] Fig. 1 shows a schematic view of a rapid test according to an embodiment of the present invention.

[0072] Unless otherwise specified or apparent from the context, the following features are represented by reference symbols: Carrier 1; Detection area 2; Sample application area 3; Control area 4; Housing 5.

[0073] In this embodiment, the carrier 1 is made of porous cellulose acetate and arranged in a housing 5. The carrier 1 comprises a detection area 2, a sample introduction area 3, and a control area 4, which are in fluid-conducting communication with each other.

[0074] Detection area 2 contains immobilized capture antibodies specific for the α1-adrenoceptor antagonist tamsulosin. Control area 4 contains immobilized tamsulosin as a control target.

[0075] The kit according to this embodiment further comprises a sample vial (not shown) containing a buffer solution with primary antibodies to which a dye is bound. In this embodiment, the dye is colloidal gold, which has a deep red color. 65387 / MB Borkenstein & Borkenstein Specialists in Ophthalmology OG, Kreuzgasse 35, 8010 Graz (AT)

[0076] The primary antibodies bind specifically to the drug, even if the drug is already bound to a capture antibody. In other words, binding of the drug to the capture antibody is possible even if the drug has already bound to the primary antibody.

[0077] The primary antibodies also bind to the control target, which is immobilized in control area 4.

[0078] The kit can now be used according to this embodiment as follows: A predetermined quantity of a sample to be examined, for example, serum from a patient treated with an α1-adrenoceptor antagonist such as tamsulosin, is placed in the sample container and mixed with the buffer solution already present in the sample container, which contains primary antibodies conjugated with the detection agent. This results in binding of the primary antibodies and the tamsulosin molecules contained in the sample.

[0079] The mixed liquid is applied to sample application area 3 of the carrier 1. Due to the capillary action of the porous material, the liquid and the substances it contains are transported towards detection area 2 and control area 4.

[0080] When the liquid reaches control area 4, any remaining free primary antibodies—that is, primary antibodies not yet bound to a tamsulosin molecule—bind to the immobilized control target. The simultaneous accumulation of colloidal gold in control area 4 results in a deep red coloration.

[0081] The liquid is then transported to detection area 2, where tamsulosin molecules present in the sample are bound by the immobilized capture antibodies. Since the tamsulosin molecules are already bound to primary antibodies and thus simultaneously to colloidal gold, gold also accumulates in detection area 2, resulting in a color change depending on the presence of tamsulosin in the sample. 65387 / MB Borkenstein & Borkenstein Specialists in Ophthalmology OG, Kreuzgasse 35, 8010 Graz (AT)

[0082] If the sample did not contain tamsulosin molecules, the detection agent would not be concentrated in detection range 2 and no red coloration would occur.

[0083] Such a kit allows for the rapid detection of whether the patient from whom the sample was taken has a relevant level of tamsulosin in their serum. This makes it possible to reduce the incidence of IFIS and, if the tamsulosin test is positive, to postpone a planned surgery or to take additional preoperative precautions to reduce risks.

Claims

65387 / MB Borkenstein & Borkenstein Specialists in Ophthalmology OG, Kreuzgasse 35, 8010 Graz (AT) Patent claims 1. Kit for the detection of an active substance that is an α1-adrenoceptor antagonist in a sample, wherein the kit comprises: - Capture antibodies designed to bind to the active ingredient, - Primary antibodies that are designed to bind to the active substance while the active substance is bound to a capture antibody, as well as - a detection agent, wherein the detection agent is associated with the primary antibody.

2. Kit according to claim 1, characterized in that the active ingredient is (R)-(-)-5-{2-[2- (2-Ethoxyphenoxy)-ethylamino]propyl}-2-methoxy-benzenesulfonamide or a pharmaceutically acceptable compound or derivative thereof.

3. Kit according to claim 1 or 2, characterized in that the capture antibodies are immobilized on a solid carrier (1 ).

4. Kit according to claim 1 or 2, characterized in that the capture antibodies are immobilized on a detection area (2) of a solid carrier (1), wherein the detection area (2) is in fluid-conducting communication with a sample delivery area (3).

5. Kit according to claim 3 or 4, characterized in that the carrier (1) is nanoparticles, in particular gold or silver nanoparticles, or that the carrier (1) comprises or is formed from a polymer material, in particular a porous one.

6. Kit according to one of claims 1 to 5, characterized in that, - that the detection agent comprises an enzyme and a detection reagent, wherein the enzyme is bound to the primary antibody, wherein the detection reagent is configured to undergo a reaction in which at least one property of the detection reagent is altered, and wherein the reaction is catalyzed by the enzyme, 65387 / MB Borkenstein & Borkenstein Specialists in Ophthalmology OG, Kreuzgasse 35, 8010 Graz (AT) and / or - that the detection agent comprises secondary antibodies, an enzyme and a detection reagent, wherein the secondary antibodies are configured to bind to a primary antibody while the latter is bound to the active substance, wherein the enzyme is bound to the secondary antibody, wherein the detection reagent is configured to undergo a reaction in which at least one property of the detection reagent is altered, and wherein the reaction is catalyzed by the enzyme.

7. Kit according to claim 6, characterized in that the detection reagent is a dye precursor, and that the property of the detection reagent which is changed in the reaction is the absorption coefficient for electromagnetic radiation in the visible and / or ultraviolet light range.

8. Kit according to any one of claims 1 to 5, characterized in that the detection agent comprises a dye that is bound to the primary antibodies or that can bind to the primary antibodies.

9. Kit according to one of claims 4 to 8, characterized in that a control area (4) is further provided on the substrate, which is in fluid-conducting communication with the sample application area (3), wherein an immobilized control substance is provided in the control area (4), wherein the control substance is configured to be bound by the primary antibodies.

10. Kit according to any one of claims 1 to 9, characterized in that the sample is a body fluid, in particular urine, serum or blood, of a subject, wherein the subject is or has been treated with a drug containing the α-adrenoceptor antagonist.

11. Kit according to one of claims 1 to 10, characterized in that the primary antibodies and the detection agent are provided in a sample vessel. 65387 / MB Borkenstein & Borkenstein Specialists in Ophthalmology OG, Kreuzgasse 35, 8010 Graz (AT) 12. Kit according to claim 11, characterized in that the sample vessel contains an aqueous buffer solution.

13. Method for detecting an active ingredient using a kit according to any one of claims 1 to 12, wherein the method comprises the following steps: - Collecting a sample, - Mixing the sample at least with the primary antibodies and the detection agent, - Reading a signal that indicates the presence of active ingredient in the sample.

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